pcr_efficiency: Calculate amplification efficiency
In pcr: Analyzing Real-Time Quantitative PCR Data

Description Usage Arguments Details Value References Examples

Uses the C_T values from a serial dilution experiment to calculate the amplification efficiency of a PCR reaction.

1	pcr_efficiency(df, amount, reference_gene, plot = FALSE)

`df`	A data.frame of C_T values with genes in the columns and samples in rows rows. Each sample are replicates of a known input/dilution given by amount
`amount`	A numeric vector of the input amounts or dilutions. The length of this vector should equal the row number of df
`reference_gene`	A character string of the column name of a control gene
`plot`	A logical (default FALSE) to indicate whether to return a data.frame or a plot

Fortunately, regardless of the method used in the analysis of qPCR data, The quality assessment are done in a similar way. It requires an experiment similar to that of calculating the standard curve. Serial dilutions of the genes of interest and controls are used as input to the reaction and different calculations are made. The amplification efficiency is approximated be the linear trend between the difference between the C_T value of a gene of interest and a control/reference (Δ C_T) and the log input amount. This piece of information is required when using the Δ Δ C_T model. Typically, the slope of the curve should be very small and the R^2 value should be very close to one. Other analysis methods are recommended when this is not the case.

When plot is FALSE returns a data.frame of 4 columns describing the line between the Δ C_T of target genes and the log of amount

gene The column names of df. reference_gene is dropped
intercept The intercept of the line
slope The slope of the line
r_squared The squared correlation

When plot is TRUE returns a graph instead shows the average and standard deviation of of the Δ C_T at different input amounts. In addition, a linear trend line is drawn.

Livak, Kenneth J, and Thomas D Schmittgen. 2001. “Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the Double Delta CT Method.” Methods 25 (4). ELSEVIER. doi:10.1006/meth.2001.1262.

# locate and read file
fl <- system.file('extdata', 'ct3.csv', package = 'pcr')
ct3 <- read.csv(fl)

# make amount/dilution variable
amount <- rep(c(1, .5, .2, .1, .05, .02, .01), each = 3)

# calculate amplification efficiency
pcr_efficiency(ct3,
               amount = amount,
               reference_gene = 'GAPDH')

# plot amplification efficiency
pcr_efficiency(ct3,
               amount = amount,
               reference_gene = 'GAPDH',
               plot = TRUE)